Poxviruses, which comprise a large family of double-stranded DNA viruses that infect vertebrate and invertebrate hosts, are distinguished by their large size, complex morphology, cytoplasmic site of replication, and encoding of proteins for viral transcription, replication, and immune defense (Moss, B. 2001 in: Fields Virology, eds. Knipe, D. M. & Howley, P. M. Lippincott Williams & Wilkins, Philadelphia, Vol. 2, pp. 2849-2883). Vaccinia virus (VAC), the best-studied member of the poxvirus family, was used for immunization against smallpox (Fenner, F. et al. 1988 Smallpox and its Eradication World Health Organization, Geneva) and subsequently as an expression vector for laboratory investigations and recombinant vaccines (Moss, B. 1996 PNAS USA 93:11341-11348). The introduction of new genes into the VAC genome is usually carried out by homologous recombination in infected cells. Typically, a foreign gene is cloned into a plasmid transfer vector downstream of a VAC promoter and flanked by VAC DNA from a non-essential site (Mackett, M. et al 1984 J Virol 49:857-864). The plasmid is then transfected into cells that are infected by VAC. Because recombination is inefficient, a variety of selection and screening methods have been devised (Earl, P. L. et al. 1998 in: Current Protocols in Molecular Biology, eds. Ausubel, F. M. et al. Greene Publishing Associates & Wiley Interscience, New York, Vol. 2, pp. 16.17.1-16.17.19). The process usually requires three to five plaque purifications over a period of several weeks. Alternative methods, involving the purification and cleavage of the VAC genome and either ligation in vitro or three-way recombination in vivo, have been described (Merchlinsky, M. & Moss, B. 1992 Virology 190:522-526; Scheiflinger, F. et al. 1992 PNAS USA 89:9977-9981; Merchlinsky, M. et al. 1997 Virology 238:444-451; Smith, E. S. et al. 2001 Nat Med 7:967-972; Timiryasova, T. M. et al. 2001 Biotechniques 31:534-540) but are cumbersome, depend on the presence of a unique restriction endonuclease site within the nearly 200,000 base pair genome, and still require plaque purification. The mutagenesis of VAC genes is even more difficult, and is usually carried out by a laborious transient dominant selection recombination scheme (Falkner, F. G. & Moss, B. 1990 J Virol 64:3108-3111).
Recently, the large DNA genomes of a baculovirus (Luckow, V. A. et al. 1993 J Virol 67:4566-4579) and several herpesviruses (Messerle, M. et al. 1997 PNAS USA 94:14759-14763; Borst, E. M. et al. 1999 J Virol 73:8320-8329; Delecluse, H. J. et al. 1998 PNAS USA 95:8245-8250; Horsburgh, B. C. et al. 1999 Gene Ther 6:922-930; Saeki, Y. et al. 1998 Hum Gene Ther 9:2787-2794; Smith, G. A. & Enquist, L. W. 1999 J Virol 73:6405-6414) have been cloned as bacterial artificial chromosomes (BACs). These circular mini-F BAC plasmids allow viral genomes to be stably maintained at low copy number and manipulated in Escherichia coli and then reconstituted as infectious virus by transfection of eukaryotic cells. The construction of baculovirus and herpesvirus BACs was relatively straightforward: plasmid sequences were inserted by recombination into the circular mature or replicating viral genomes and the latter were propagated in E. coli. Poxvirus genomes, however, are composed of linear double-stranded DNA molecules with covalently closed hairpin ends (Baroudy, B. M. et al. 1982 Cell 28:315-324) that are resolved from transient head-to-head or tail-to-tail concatemers during replication (Moyer, R. W. & Graves, R. L. 1981 Cell 27:391-401; Baroudy, B. M. et al. 1982 Cold Spring Harbor Symp Quant Biol 47:723-729; Merchlinsky, M. et al. 1988 J Mol Biol 199:399-413). Therefore, the method used for cloning baculovirus and herpesvirus genomes seemed inapplicable.